Hepatocyte transdifferentiation of into functional insulin-producing cells (IPCs) by transcription factor-(TF-) mediated genetic reprogramming is a promising option for cell replacement and gene therapy, one that exploits the common embryonal origin of liver and pancreas cells. In Specific Aim-1, we propose to investigate the molecular mechanisms of the liver-to-endocrine-pancreas transdifferentiation using gene knock-in or knockout strategies, and we will determine the most effective combination of a-cell TFs with external factors in controlling the process of the transdifferentiation using stably transfected hepatic cell lines. Hypotheses to be tested: (1) Pdx1 expression alone is insufficient for selective transdifferentiation of hepatic cells into endocrine pancreatic IPCs; and (2) Additional factors including other TFs downstream of Pdx1 and external factors are needed to reprogram hepatocytes into the pathway of pancreatic IPCs. In Specific Aim-2, we propose to determine factors critical for an in vivo selective transdifferentiation of liver cells into IPCs in mice using a hydrodynamic-based tail vein delivery system, portal vein infusion, or direct intrahepatic injection. Hypotheses to be tested: (1) The in vivo diabetic microenvironment plays a permissive role in facilitating Pdx1 protein in the hepatocytes to activate insulin gene expression to produce insulin, and in turn, reducing blood glucose level; (2) Pdx1 expression alone (even its modified form Pdxl-VP16) in the hepatocytes, may not be sufficient to specify the pancreatic endocrine differentiation due to the lack of appropriate protein partners such as other TFs related to pancreatic endocrine development; and (3)The short lifespan of the insulin-producing cells in the liver may result from the transfection of terminally differentiated hepatocytes by Pdx1gene injection, while transfection/transduction of hepatic stem cells with target genes may produce long lasting effects of reversing hyperglycemia in diabetic mice. In Specific Aim-3, we propose to transdiferentiate primary hepatocytes into IPCs to learn whether the liver-derived IPCs can escape the autoimmune attack in nonobese diabetic (NOD) type 1 diabetes (T1D) mouse model. Hypothesis to be tested: Primary hepatocytes and hepatic stem oval cells can be converted into IPCs by genetic modifications, and these liver-derived-genetically modified-IPCs may reduce or prevent the autoimmune destruction and reverse T1D in NOD animal models.